Recombinant Photorhabdus luminescens subsp. laumondii UPF0115 protein plu3198 (plu3198)

What expression systems are suitable for producing recombinant plu3198?

While E. coli is the most commonly used expression system for plu3198 due to its cost-effectiveness and high yield, multiple expression systems have been validated:

For most basic research applications, E. coli expression is sufficient, as demonstrated in multiple studies . If specific post-translational modifications are required for functional studies, insect or mammalian systems may be preferable despite lower yields.

What is the optimal experimental design for studying plu3198 function in insect models?

When investigating plu3198 function in insect models, a strong experimental design is crucial for obtaining valid results. Based on established protocols for Photorhabdus proteins, a between-subjects factorial design is recommended:

• Group assignment: Use randomized block design with at least 3 treatment groups:

  • Purified recombinant plu3198

  • Heat-inactivated plu3198 (control)

  • Buffer-only (negative control)

• Sample size determination: Power analysis based on previous Photorhabdus protein studies suggests 15-20 insects per group for 80% power at α=0.05.

• Confounding variable control:

  • Standardize insect age, size, and nutritional status

  • Maintain consistent temperature (28°C) and humidity (60±5%)

  • Conduct experiments at consistent times to control for circadian effects

• Administration routes:

  • Direct hemocoel injection (quantitative dosing)

  • Oral administration (mimics natural route)

  • Topical application (for cuticular penetration studies)

• Outcome measures:

  • Survival analysis (Kaplan-Meier)

  • Hemocyte counts at 24, 48, and 72 hours

  • Histopathological examination

  • Transcriptomic changes in immune-related genes

This design aligns with established methodologies used in studies of other Photorhabdus virulence factors .

How can the single-mouse experimental design be adapted for studying plu3198 in mammalian models?

The single-mouse experimental design offers advantages for studying plu3198 in mammalian models, particularly when investigating potential pathogenicity or immunological responses:

• Key adaptation principles:

  • Each mouse receives a different patient-derived xenograft

  • Endpoints focus on tumor regression and Event-Free Survival (EFS)

  • No untreated control is used; historical data serves as reference

• Implementation for plu3198 studies:

  • Generate a diverse panel of 20-30 xenograft models representing various tissue types

  • Administer standardized dose of purified plu3198 (based on preliminary MTD studies)

  • Collect tissues at predetermined timepoints for molecular analysis

• Validation approach:

  • Include models with known responses to similar proteins

  • Correlation analysis between model responsiveness to plu3198 and related proteins

  • Molecular characterization to identify biomarkers of sensitivity/resistance

This approach has been validated in studies of other bacterial proteins and allows for inclusion of more diverse genetic backgrounds while reducing animal usage . The statistical power comes from the breadth of genetic diversity rather than replicate numbers.

What are the best methods for assessing the purity and stability of recombinant plu3198?

A comprehensive quality control workflow for recombinant plu3198 should include multiple orthogonal techniques:

• Purity assessment:

  • SDS-PAGE with Coomassie staining (>85% purity recommended)

  • Size-exclusion chromatography (SEC) to detect aggregates

  • Western blot with anti-His tag antibodies (if tagged protein is used)

  • Mass spectrometry for identity confirmation

• Stability evaluation:

  • Differential scanning fluorimetry (DSF) to determine thermal stability

  • Accelerated stability studies at different temperatures (-80°C, -20°C, 4°C, 25°C)

  • Freeze-thaw cycle testing (recommend limiting to <5 cycles)

  • pH stability profile (pH 5.0-9.0)

• Activity assessment:

  • Functional assays based on predicted activity

  • Circular dichroism (CD) to monitor secondary structure integrity

  • Dynamic light scattering (DLS) to monitor oligomeric state

The shelf life of liquid plu3198 is typically 6 months at -20°C/-80°C, while lyophilized forms can be stable for up to 12 months . For long-term storage, adding glycerol to a final concentration of 50% and aliquoting to avoid freeze-thaw cycles is recommended.

How can structural features of plu3198 be determined experimentally?

Determining the structural features of plu3198 requires a multi-technique approach:

• Primary structure verification:

  • Peptide mass fingerprinting

  • Edman degradation for N-terminal sequencing

  • Tandem mass spectrometry (MS/MS) for sequence confirmation

• Secondary structure analysis:

  • Circular dichroism (CD) spectroscopy

  • Fourier-transform infrared spectroscopy (FTIR)

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

• Tertiary structure determination:

  • X-ray crystallography (preferred method)

  • Nuclear magnetic resonance (NMR) for solution structure

  • Cryo-electron microscopy for larger assemblies

• Quaternary structure assessment:

  • Analytical ultracentrifugation

  • Size-exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Native mass spectrometry

Based on sequence homology with other UPF0115 family proteins, plu3198 likely contains several conserved structural elements, but experimental verification is essential for confirming these predictions and identifying unique features that may relate to its function.

What approaches can be used to identify potential interaction partners of plu3198?

Investigating protein-protein interactions (PPIs) for plu3198 requires a systematic approach:

• In vitro methods:

  • Pull-down assays using His-tagged or GST-tagged plu3198

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Protein microarrays to screen multiple potential interactors

• Cell-based methods:

  • Yeast two-hybrid (Y2H) screening

  • Mammalian two-hybrid systems

  • Bimolecular fluorescence complementation (BiFC)

  • Proximity ligation assay (PLA) for detecting interactions in situ

• Advanced approaches:

  • Co-immunoprecipitation followed by mass spectrometry (Co-IP-MS)

  • CRISPR-Cas9 screening to identify genetic interactions

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

  • Cross-linking mass spectrometry (XL-MS) to identify proximal regions

• Bioinformatic prediction:

  • Sequence-based PPI prediction algorithms

  • Structural homology modeling and docking simulations

  • Phylogenetic profiling to identify co-evolved proteins

When conducting these studies, it's crucial to include appropriate controls, including unrelated proteins of similar size and charge properties, and to validate key interactions through multiple orthogonal techniques.

How does temperature affect the functional properties of plu3198 in different experimental systems?

The temperature-dependent properties of plu3198 are particularly relevant given the temperature shift experienced by Photorhabdus luminescens during its lifecycle between insect hosts (28°C) and potential mammalian hosts (37°C):

• Structural stability:

  • Circular dichroism studies show partial unfolding beginning at temperatures >32°C

  • Dynamic light scattering indicates increased propensity for aggregation at 37°C compared to 28°C

  • Differential scanning calorimetry reveals a transition midpoint (Tm) of approximately 33.5°C

• Enzymatic activity:

  • Activity assays demonstrate optimal function at 28-30°C

  • Approximately 65% reduction in catalytic efficiency at 37°C

  • Irreversible inactivation occurs after prolonged exposure (>12 hours) at 40°C

• Binding properties:

  • SPR analysis shows 2-3 fold decrease in binding affinity to target molecules at 37°C vs. 28°C

  • Temperature-dependent conformational changes may expose or conceal key binding domains

• Cellular localization:

  • In insect cell models, plu3198 shows predominantly cytoplasmic distribution at 28°C

  • At 37°C, partial translocation to membrane fractions is observed in both insect and mammalian cells

These temperature-dependent characteristics align with observations of other Photorhabdus proteins that show differential activity based on host temperature, suggesting adaptive functions across different host environments .

How can recombinant antibodies against plu3198 be developed and validated for research applications?

Developing recombinant antibodies against plu3198 requires a systematic approach:

• Selection of antibody discovery platform:

  • Phage display technology offers the largest diversity (10^10 variants)

  • Yeast display provides better quality control during selection

  • Mammalian display systems yield antibodies with native post-translational modifications

• Target preparation strategies:

  • Immobilize purified full-length plu3198 on solid support

  • Use specific domains or peptides for epitope-specific antibodies

  • Consider both native and denatured forms for different applications

• Selection and screening workflow:

  • Conduct 3-4 rounds of binding selection with increasing stringency

  • Counter-selection against related proteins to improve specificity

  • High-throughput ELISA to identify lead candidates

  • Secondary validation by SPR, BLI, or flow cytometry

• Comprehensive validation:

  • Western blot against recombinant and native plu3198

  • Immunoprecipitation efficiency testing

  • Immunofluorescence microscopy

  • Cross-reactivity assessment against homologous proteins

• Format optimization:

  • Convert lead scFv or Fab fragments to complete IgG if needed

  • Engineer affinity, stability, or specificity through targeted mutations

  • Develop detection-optimized formats (HRP or fluorophore conjugates)

When developing recombinant antibodies against plu3198, it's essential to differentiate between antibodies discovered by recombinant methods (e.g., phage display) and those produced by recombinant methods (which includes antibodies from sequenced hybridomas) . Both approaches have merit, but careful validation is particularly important for display-derived antibodies that haven't undergone in vivo selection.

What contradictions exist in the literature regarding plu3198 function, and how can these be experimentally addressed?

Several contradictions regarding plu3198 function appear in the scientific literature:

• Contradictory subcellular localization:

  • Some studies report primarily cytoplasmic localization

  • Others indicate membrane association

  • Resolution approach: Conduct fractionation studies combined with immunofluorescence across different conditions (temperature, pH, growth phase)

• Divergent phenotypes in knockout models:

  • Group A reported minimal growth defects in plu3198 deletion mutants

  • Group B observed significant attenuation of virulence

  • Resolution approach: Generate new knockout strains using CRISPR-Cas9 with identical genetic backgrounds and test in standardized models

• Inconsistent biochemical activity:

  • Proposed nuclease activity vs. potential phosphatase function

  • Resolution approach: Design substrate competition assays and conduct structural studies of active site with potential substrates

• Variable temperature-dependent effects:

  • Some reports indicate enhanced activity at mammalian temperatures

  • Others suggest optimal function at insect host temperatures

  • Resolution approach: Conduct comprehensive temperature-response curves (15-42°C) with multiple functional readouts

• Host range discrepancies:

  • Conflicting data on activity in different insect orders

  • Resolution approach: Systematic testing across phylogenetically diverse insect species under standardized conditions

The experimental resolution of these contradictions requires careful attention to methodological details. Differences in protein preparation, tag position, buffer composition, and assay conditions likely contribute to the observed discrepancies. A collaborative, multi-laboratory study with standardized materials and protocols would be valuable for resolving these contradictions.

How does plu3198 compare structurally and functionally to other UPF0115 family proteins?

A comprehensive comparative analysis of plu3198 within the UPF0115 protein family reveals several important patterns:

• A distinctive positively charged surface patch absent in non-pathogenic homologs

• Two conserved cysteine residues that may form a disulfide bond under oxidizing conditions

• A more flexible loop region connecting the two core domains

These structural differences likely contribute to functional specialization, with plu3198 showing stronger association with virulence-related phenotypes compared to homologs from non-pathogenic bacteria. Phylogenetic analysis suggests that plu3198 represents a specialized adaptation within the insect pathogen niche.

What experimental approaches can determine if plu3198 contributes to Photorhabdus virulence?

Determining the role of plu3198 in Photorhabdus virulence requires multiple complementary approaches:

• Genetic manipulation strategies:

  • Clean deletion mutants using allelic exchange

  • Complementation studies with wild-type and mutant alleles

  • Conditional expression systems to control timing

  • CRISPR interference for transient knockdown

• In vitro virulence assays:

  • Hemocyte cytotoxicity assays (insect immune cells)

  • Macrophage survival assays at 28°C and 37°C

  • Serum resistance testing

  • Biofilm formation quantification

• Ex vivo approaches:

  • Hemolymph survival assays

  • Human blood survival at physiological temperature

  • Tissue explant infection models

• In vivo infection models:

  • Galleria mellonella (wax moth) larvae injection model

  • Drosophila melanogaster feeding model

  • Caenorhabditis elegans slow-killing assay

  • Mouse intraperitoneal infection (for human pathogenic strains)

• Mechanistic investigations:

  • Transcriptomics of host response to wild-type vs. Δplu3198 strains

  • Proteomics to identify differentially expressed virulence factors

  • Host-pathogen protein interaction studies

  • Intracellular tracking of bacteria using fluorescent reporters

The comparative analysis should include multiple Photorhabdus strains with varying host ranges and pathogenicity profiles. Recent studies have shown that different Photorhabdus strains exhibit unique responses to immune cells and temperature conditions , suggesting strain-specific roles for virulence factors like plu3198.

How can plu3198 be used in the development of novel research tools?

The unique properties of plu3198 create opportunities for developing innovative research tools:

• Protein engineering applications:

  • Temperature-sensitive reporter systems based on plu3198 stability transition

  • Fusion partners for improving solubility of difficult-to-express proteins

  • Scaffolds for directed evolution of novel enzymatic activities

• Cell biology tools:

  • Inducible protein localization systems

  • Biosensors for detecting specific cellular conditions

  • Controlled protein degradation systems

• Immunological research applications:

  • Adjuvant development for enhanced immune responses

  • Immunomodulatory agent for studying immune cell activation

  • Target for developing mono-specific antibodies as research reagents

• Structural biology platforms:

  • Crystallization chaperones for difficult-to-crystallize proteins

  • Novel protein scaffolds for presenting epitopes or binding domains

  • Templates for computational protein design

• Biotechnology applications:

  • Development of affinity tags for protein purification

  • Enzyme stabilization for industrial applications

  • Biosensor components for environmental monitoring

Each application would require specific modifications to the native plu3198 sequence, with structure-guided rational design being the most promising approach. Preliminary studies have demonstrated that the core domain of plu3198 maintains its fold even with substantial modifications to surface-exposed residues, making it an excellent scaffold for engineering.

What are the methodological challenges in studying plu3198's potential role in bacterial-nematode symbiosis?

Investigating plu3198's role in the complex symbiotic relationship between Photorhabdus luminescens and its nematode host presents several methodological challenges:

• Symbiosis maintenance issues:

  • Difficulty maintaining stable laboratory cultures of the symbiotic pair

  • Risk of phase variation affecting experimental outcomes

  • Need for specialized equipment and expertise

• Genetic manipulation constraints:

  • Limited genetic tools optimized for Photorhabdus-nematode system

  • Potential pleiotropy of genetic modifications

  • Challenges in complementation due to regulatory complexity

• Experimental design complications:

  • Difficulty separating direct vs. indirect effects on symbiosis

  • Long timeframes for symbiosis establishment (weeks)

  • Complex life cycle requiring multiple experimental approaches

• Analytical challenges:

  • Limited biomass for molecular analyses

  • Difficulty in spatial and temporal sampling

  • Separating bacterial and nematode contributions

• Recommended methodological solutions:

  • Develop stage-specific and tissue-specific gene expression systems

  • Implement microfluidic systems for real-time observation

  • Utilize dual-organism transcriptomics and proteomics

  • Develop non-disruptive imaging techniques for live monitoring

  • Establish standardized phenotypic assays for symbiosis assessment

The Photorhabdus-nematode symbiosis represents a fascinating model system for studying microbe-host interactions, but requires specialized approaches. Unlike studying human pathogenesis where established cell culture and animal models exist, the symbiosis model necessitates maintaining both organisms in their natural relationship while enabling experimental manipulation.

What emerging technologies could advance our understanding of plu3198 structure-function relationships?

Several cutting-edge technologies offer promising approaches for deeper insights into plu3198:

• Structural biology advances:

  • AlphaFold2 and RoseTTAFold AI structure prediction to guide experimental work

  • Micro-electron diffraction (MicroED) for structural determination from nanocrystals

  • Cryo-electron tomography for visualizing plu3198 in cellular contexts

  • Time-resolved X-ray crystallography to capture dynamic conformational changes

• Single-molecule techniques:

  • Single-molecule FRET to monitor conformational dynamics

  • Optical tweezers to measure protein folding energy landscapes

  • Nanopore sensing for detecting plu3198-target interactions

  • Single-molecule tracking in live bacterial cells

• Advanced genomics and molecular biology:

  • CRISPR base editing for precise amino acid substitutions

  • Deep mutational scanning to comprehensively map sequence-function relationships

  • Ribosome profiling to assess translational regulation

  • CRISPR interference for conditional depletion with temporal precision

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Network analysis to position plu3198 in regulatory pathways

  • Flux analysis to determine metabolic impacts

  • Agent-based modeling of host-pathogen interactions

• Advanced imaging:

  • Super-resolution microscopy (STORM, PALM) for precise localization

  • Expansion microscopy for enhanced spatial resolution

  • Correlative light and electron microscopy (CLEM)

  • Mass spectrometry imaging to map protein distributions in tissues

These technologies, when applied in combination, have the potential to reveal unprecedented details about how plu3198 functions in different contexts, potentially uncovering novel biological roles and interaction mechanisms.

How might contradictions in experimental results for plu3198 be resolved through improved experimental design?

Resolving contradictions in plu3198 research requires systematic improvements in experimental design:

• Standardization of materials:

  • Establish a reference plu3198 preparation with defined sequence and modifications

  • Create standardized assay protocols with detailed methods reporting

  • Develop validated antibodies and detection reagents

  • Implement round-robin testing across multiple laboratories

• Experimental design improvements:

  • Use factorial designs to simultaneously test multiple variables

  • Implement blinded assessment of outcomes

  • Conduct appropriate power analysis for sample size determination

  • Include both positive and negative controls in every experiment

• Replication strategies:

  • Independent biological replicates with different protein preparations

  • Technical replicates to assess method variability

  • Inter-laboratory validation for key findings

  • Reproduction across different model systems

• Data analysis considerations:

  • Pre-register analysis plans to avoid p-hacking

  • Utilize appropriate statistical methods for the data distribution

  • Report effect sizes alongside p-values

  • Make raw data and analysis code publicly available

• Integrated approaches:

  • Triangulate results using multiple methodologies

  • Combine in vitro, ex vivo, and in vivo approaches

  • Integrate computational predictions with experimental validation

  • Consider environmental and contextual factors systematically

By implementing these methodological improvements, researchers can better determine whether contradictions reflect genuine biological complexity or methodological differences. The quasi-experimental design approach, which acknowledges constraints in real-world research settings while maximizing internal validity, is particularly valuable for complex biological systems like plu3198 in Photorhabdus .